Although iron and copper are essential nutrients, the pathological processes associated with the various forms of metal overload demonstrate that these metals can become toxic when high exposures occur. Examples of occupational poisoning are still reported but dominant interest now lies in the possibility of insidious effects resulting from long term exposures. Cellular injury from toxic metals may occur by a number of molecular mechanisms including metal-induced formation of free radicals. The most direct technique available for the detection of free radicals is ESR spectroscopy which for use in living systems requires a spin- trapping approach. In the study of free radical formation in vivo, we utilized the scavenging reaction in which the hydroxyl radical is converted into the methyl radical via its reaction with dimethyl sulfoxide (DMSO). The methyl radical is them detected as its long-lived phenyl N-t-butylnitrone (PBN) adduct. All of the previous studies used doses of the metals near their LD50 levels. Significantly lower doses did not give detectable radical adduct spectra. Nevertheless, we attempted to detect hydroxyl radical generation in rats with chronic dietary iron loading. Rats were fed a diet modified with 1255 mg/kg iron as ferric citrate. After 10 weeks the rats were given an injection of PBN dissolved in DMSO, and the PBN/CH3 radical adduct was detected in the bile. This was the first evidence of hydroxyl radical generation in chronic iron loaded rats. In addition, we showed that free radical generation occurred in rats when the hepatic iron concentration was relatively low and there was no detectable liver damage. We have also demonstrated experimental procedures necessary to inhibit ex vivo metal ion-induced free radical chemistry in cases of metal overload.